1. Field of the Invention
The present invention relates to a novel class of artemisinin dimers which demonstrate useful anticancer activity.
2. Description of the State of Art
Artemisia annua L., also known as qing hao or sweet wormwood, is a pervasive weed that has been used for many centuries in Chinese traditional medicine as a treatment for fever and malaria. Its earliest mention, for use in hemorrhoids, occurs in the Recipes for 52 Kinds of Diseases found in the Mawangdui Han dynasty tomb dating from 168 B.C. Nearly five hundred years later Ge Hong wrote the Zhou Hou Bei Ji Fang Handbook of Prescriptions for Emergency Treatments) in which he advised that a water extract of qing hao was effective at reducing fevers. In 1596, Li Shizhen, the famous herbalist, wrote that chills and fever of malaria can be combated by qing hao preparations. Finally, in 1971, Chinese chemists isolated from the leafy portions of the plant the substance responsible for its reputed medicinal action. This crystalline compound, called qinghaosu, also referred to as QHS or artemisinin, is a sesquiterpene lactone with an internal peroxide linkage.
Artemisinin (3,6,9-trimethyl-9,10b-epi-dioxyperhydropyranol 4,3,2-jk!benzoxepin-2-one) is a member of the amorphane subgroup of cadinenes and has the following structure (I). ##STR2## Artemisinin or QHS was the subject of a 1979 study conducted by the Qinghaosu Antimalarial Coordinating Research Group involving the treatment of 2099 cases of malaria (Plasmodium vivax and Plasmodium falciparum in a ratio of about 3:1) with different dosage forms of QHS, leading to the clinical cure of all patients. See, Qinghaosu Antimalarial Coordinating Research Group, Chin. Med. J., 92:811 (1979). Since that time QHS has been used successfully in several thousand malaria patients throughout the world including those infected with both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum. Assay of QHS against P. falciparum, in vitro, revealed that its potency is comparable to that of chloroquine in two Hanian strains (Z. Ye, et al., J. Trad. Chin. Med., 3:95 (1983)) and of mefloquine in the Camp (chloroquine-susceptible) and Smith (chloroquine-resistant) strains, D. L. Klayman, et al., J. Nat. Prod., 47:715 (1984).
Most research suggests that QHS acts by an oxidative mechanism and it effects changes in both red blood cells and in the limiting and other membranes of the malarial parasite. At concentrations much higher than those used clinically, QHS affects red blood cell deformability in a manner which suggests that QHS acts as an efficient prooxidant, M. D. Scott, et al., J. Lab. Clin. Med., 114:40 (1989). At even higher concentrations QHS brings about complete lysis of red blood cells, G. Haoming, Zhongguo Yaoli Xuebao, 7:269 (1986). The mechanism of action of QHS appears to involve two steps, S. Meshnick, Transactions of the Royal Society of Tropical Medicine and Hygiene 88(1): S1/31-S1/32, (1994). In the first step, activation, intra-parasite iron catalyzes the cleavage of the endoperoxide bridge and the generation of free radicals. A free radical is a short-lived and highly reactive molecule that contains an unpaired electron. In the second step, alkylation, the QHS-derived free radical forms covalent bonds with parasitic proteins which leads to alternations in ribosomal organization and the endoplasmic reticulum. Nuclear membrane blebbing develops followed by segregation of the nucleoplasm. The parasite continues to undergo degenerative changes with disorganization and death occurring from eight hours onwards following the initial exposure to QHS.
Although QHS is effective at suppressing the parasitemias of P. vivax and P. falciparum, the problems encountered with recrudescence, and the compounds solubility, due to the lactone ring in QHS, led scientists to modify QHS chemically, a difficult task because of the chemical reactivity of the peroxide linkage which is an essential moiety for antimalarial activity.
Reduction of QHS in the presence of sodium borohydride results in the production of dihydroartemesinin (II-1) or DHQHS, (illustrated in structure II below), in which the lactone group is converted to a lactol hemiacetal) function, with properties similar to QHS. QHS in methanol is reduced with sodium borohydride to an equilibrium mixture of .alpha.- and .beta.-isomers of dihydroartemisinin. The yield under controlled conditions is 79% (QHS, 0.85M with NaBH.sub.4 6.34M. 7.5 equivalents in methanol, 12 L at 0.degree.-5.degree. C. for about 3 hours followed by quenching to neutrality at 0.degree.-5.degree. C. and dilution with water to precipitate dihydroartemisinin), A. Brossi, et al., Journal of Medicinal Chemistry, 31:645-650 (1988). Using DHQHS as a starting compound a large number of other derivatives, such as, ##STR3## artemether (compound II-2), arteether (II-3), sodium artesunate (II-4), artelinic acid (II-5), sodium artelinate (II-6), DHQHS condensation by-product (II-7) and the olefinic compound, structure III, ##STR4## have been produced.
Artemether (II-2) is produced by reacting .beta.-DHQHS with boron trifluoride (BF.sub.3) etherate or HCl methanol:benzene (1:2) at room temperature. A mixture of .beta.- and .alpha.-artemether (70:30) is obtained, from which the former is isolated by column chromatography and recrystallized from hexane or methanol, R. Hynes, Transactions of the Royal Society of Tropical Medicines and Hygiene, 88(1): S1/23-S1/26 (1994). For arteether (II-3), (Brossi, et al., 1988), the .alpha.-isomer is equilibrated (epimerized) to the .beta.-isomer in methanol:benzene mixture containing BF.sub.3 etherate. Treatment of DHQHS with an unspecified dehydrating agent yields both the olefinic compound, (III), and the DHQHS condensation by-product (II-7), formed on addition of DHQHS to (III), M. Cao, et al., Chem. Abstr., 100:34720k (1984). Until recently, the secondary hydroxy group in DHQHS (1I-1) provided the only site in an active QHS related compound that had been used for derivatization. See B. Venugopalan "Synthesis of a Novel Ring Contracted Artemisinin Derivative," Bioorganic & Medicinal Chemistry Letters, 4(5):751-752 (1994).
The potency of various QHS-derivatives in comparison to QHS as a function of the concentration at which the parasitemia is 90 percent suppressed (SD.sub.50) was recently reported by D. L. Klayman, "Qinghaosu (Artemisinin): An Antimalarial Drug from China," Science 228:1049-1055 (1985). Dr. Klayman found that the olefinic compound III is inactive against P. bergher-infected mice, whereas, the DHQHS condensation by-product (II-7) has an SD.sub.50 of 10 mg/Kg in P. bergher-infected mice. Thus, the DHQHS ether dimer proved to be less potent than QHS, which has an SD.sub.50 of 6.20 mg/Kg. Following, in order of their overall antimalarial efficacy, are the three types of derivatives of DHQHS (II-1) that have been produced: (QHS)&lt;ethers (II, R=alkyl)&lt;esters II, R.dbd.C(.dbd.O)-alkyl or -aryl!&lt;carbonates II, R.dbd.C(.dbd.O)O-alkyl or -aryl!.
Over the past twenty years only a few drugs isolated from higher plants have yielded clinical agents, the outstanding examples being vinblastine and vincristine from the Madagascan periwinkle, Catharanthus roseus, etoposide, the semi-synthetic lignam, from May-apple Podophyllum peltatum and the diterpenoid taxol, commonly referred to as paclitaxel, from the Pacific yew, Taxus brevifolia. Of these agents, paclitaxel is the most exciting, recently receiving approval by the Food and Drug Administration for the treatment of refractory ovarian cancer. Since the isolation of QHS, there has been a concerted effort by investigators to study other therapeutic applications of QHS and its derivatives.
National Institutes of Health reported that QHS is inactive against P388 leukemia. See NCI Report on NSC 369397 (tested on 25 Oct. 1983). Later anticancer studies that have been conducted on cell line panels consisting of 60 lines organized into nine, disease-related subpanels including leukemia, non-small-cell lung cancer, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers, further confirm that QHS displays very little anticancer activity. A series of artemisinin-related endoperoxides were tested for cytoxicity to Ehrlich ascites tumor (EAT) cells using the microculture tetrazolum (MTT) assay, H. J. Woerdenbag, et al. "Cytotoxicity of Artemisinin-Related Endoperoxides to Ehrlich Ascites Tumor Cells," Journal of Natural Products, 56(6):849-856 (1993). The MTT assay, used to test the artemisinin-related endoperoxides for cytoxicity, is based on the metabolic reduction of soluble tetrazolium salts into insoluble colored formazan products by mitochondrial dehydrogenase activity of the tumor cells. As parameters for cytoxicity, the IC.sub.50 and IC.sub.80 values, the drug concentrations causing respectively 50% and 80% growth inhibition of the tumor cells, were used. QHS (I), had an IC.sub.50 value of 29.8 .mu.M. Derivatives of DHQHS (II-1) being developed as antimalarial drugs (artemether (II-2), arteether (III-3), sodium artesunate (II-4), artelinic acid (II-5) and sodium artelinate (II-6)), exhibited a somewhat more potent cytoxicity. Their IC.sub.50 values ranged from 12.2 .mu.M to 19.9 .mu.M. The DHQHS condensation by-product (II-7), disclosed previously by M. Cao, et al., 1984, was the most potent cytotoxic agent, its IC.sub.50 being 1.4 .mu.M. At this drug concentration, the condensation by-product (II-7) is approximately twenty-two times more cytoxic than QHS and sixty times more cytotoxic than DHQHS (II-1), the parent compound.
There is still a need, therefore, for developing structural analogs of QHS as antitumor agents that have potency equivalent to or greater than known anticancer agents.